J. Phycol. 42, 482–492 (2006) r 2006 Phycological Society of America DOI: 10.1111/j.1529-8817.2006.00210.x DEFINING THE MAJOR LINEAGES OF RED ALGAE (RHODOPHYTA)1 Hwan Su Yoon Department of Biological Sciences and Roy J. Carver Center for Comparative Genomics, University of Iowa, 468 Biology Building, Iowa City, Iowa 52242, USA Kirsten M. Mu¨ller Department of Biology, University of Waterloo, Waterloo, ON, Canada N2L 3G1 Robert G. Sheath Office of the Provost, California State University San Marcos, San Marcos, California 92096, USA Franklyn D. Ott 905 NE Hilltop Drive, Topeka, Kansas 66617, USA and Debashish Bhattacharya2 Department of Biological Sciences and Roy J. Carver Center for Comparative Genomics, University of Iowa, 446 Biology Building, Iowa City, Iowa 52242, USA Previous phylogenetic studies of the Rhodophyta dospora,andRufusia). We also describe a new have provided a framework for understanding red order, Rhodellales, and a new family, Rhodellaceae algal phylogeny, but there still exists the need for a (with Rhodella, Dixoniella, and Glaucosphaera). comprehensive analysis using a broad sampling of Key index words: Bangiophyceae; Compsopogono- taxa and sufficient phylogenetic information to phyceae; Cyanidiophyceae; Florideophyceae; clearly define the major lineages. In this study, we Porphyridiophycae; red algal lineages; Rhod- determined 48 sequences of the PSI P700 chl a ellophyceae; Rhodophyta; Stylonematophyceae apoprotein A1 (psaA) and rbcL coding regions and established a robust red algal phylogeny to identify Abbreviations: BPP, Bayesian posterior probabili- the major clades. The tree included most of the lin- ties; ML, maximum likelihood; MP, maximum par- eages of the Bangiophyceae (25 genera, 48 taxa). simony; PsaA, PSI P700 chlorophyll a apoprotein Seven well-supported lineages were identified with A1; PsaB, PSI P700 chlorophyll a apoprotein A2; this analysis with the Cyanidiales having the earliest PsbA, PSII reaction center protein D1; PsbC, PSII divergence and being distinct from the remaining 44 KD apoprotein; PsbD, PSII D2 reaction center taxa; i.e. the Porphyridiales 1–3, Bangiales, Flori- protein; TBR, tree bisection-reconnection deophyceae, and Compsopogonales. We also analy- zed data sets with fewer taxa but using seven pro- teins or the DNA sequence from nine genes to resolve inter-clade relationships. Based on all of The red algae (Rhodophyta) are a distinct eukaryotic these analyses, we propose that the Rhodophyta lineage whose members are united in phylogenetic anal- contains two new subphyla, the Cyanidiophytina yses of nuclear, plastid, and mitochondrial genes (Fresh- with a single class, the Cyanidiophyceae, and the water et al. 1994, Ragan et al. 1994, Van de Peer and De Rhodophytina with six classes, the Bangiophyceae, Wachter 1997, Burger et al. 1999, Yoon et al. 2002b, Compsopogonophyceae, Florideophyceae, Por- 2004). Rhodophytes lack chl b and c but contain all- phyridiophyceae classis nov. (which contains Por- ophycocyanin, phycocyanin, and phycoerythrin in the phyridium, Flintiella,andErythrolobus), Rhod- form of phycobilisomes on unstacked thylakoids. The ellophyceae, and Stylonematophyceae classis nov. plastid in these taxa is bound by two membranes and (which contains Stylonema, Bangiopsis, Chroodacty- produces floridean starch that is deposited in the cyto- lon, Chroothece, Purpureofilum, Rhodosorus, Rho- plasm. All members of this group lack flagella and cent- rioles in all stages of the life history (Gabrielson et al. 1990, Graham and Wilcox 2000). It is believed that the 1Received 13 September 2005. Accepted 27 December 2005. red algal plastid originated from a cyanobacterial pri- 2Author for correspondence: e-mail debashi-bhattacharya@ mary endosymbiosis and this organelle shares a com- uiowa.edu. monancestrywithgreenandglaucophytealgae 482 THE MAJOR LINEAGES OF RED ALGAE 483 (Bhattacharya and Medlin 1995, Delwiche et al. 1995, MATERIALS AND METHODS Cavalier-Smith 1998, McFadden 1999, Bhattacharya et al. 2004, Rodrı´guez-Ezpeleta et al. 2005). These three Taxon sampling and sequencing. Forty-eight red algal taxa ‘‘primary’’ plastid-containing groups are considered to were used to infer the phylogeny of the Bangiophyceae (Ta- be taxonomically united in the kingdom Plantae (Cava- ble 1). The data set included all bangiophycean orders and 25 genera from the different phylogenetic lineages (Garbary lier-Smith 1998) or Archaeplastida (Adl et al. 2005). and Gabrielson 1990, Mu¨ller et al. 2001). Our alignment also Traditionally, the phylum Rhodophyta has been di- included 10 green algae, two glaucophytes, and three cyano- vided into two classes (or subclasses), Bangiophyceae bacteria as the outgroup (Bhattacharya and Medlin 1995, (Bangiophycidae) and Florideophyceae (Florideo- Moreira et al. 2000). We obtained algal cultures from the phycidae) (Garbary and Gabrielson 1990). However, Culture Collection of Algae & Protozoa (CCAP), Provasoli- recent studies have concluded that the Florideo- Guillard National Center for Culture of Marine Phytoplank- phyceae form a monophyletic group with the order ton (CCMP), the Dipartimento di Biologia Vegetale (DBV) culture collection at the University of Naples, the Sammlung Bangiales (Oliveira and Bhattacharya 2000, Mu¨ller von Algenkulturen (SAG) at the University of Go¨ttingen, and et al. 2001, Yoon et al. 2002b, Saunders and Hommer- the Culture Collection of Algae at the University of Texas at sand 2004). The Bangiophyceae, which has in the past Austin (UTEX). Some of the bangiophytes were collected in been divided into six orders (Bangiales, Cyanidiales, the field and/or maintained in the private collection of F. D. Ott. Compsopogonales, Erythropeltidales, Porphyridiales, The algal cells were frozen in liquid nitrogen and ground t and Rhodochaetales), is now considered to form a se- with glass beads using a glass rod and/or Mini-BeadBeater (Biospec Products Inc., Bartlesville, OK, USA). Total genomic ries of radiations that define the ancestral lineages of DNA was extracted using the DNeasy Plant Mini Kit (Qiagen, the red algae (Gabrielson et al. 1985, Freshwater et al. Santa Clarita, CA, USA). PCR were carried out using specific 1994, Ragan et al. 1994). Comprehensive phylogenetic primers for each plastid gene (Yoon et al. 2002a). Because in- studies of the Bangiophyceae (Oliveira and Bhatta- trons were found in the psaA gene of some red algae, the RT- charya 2000, Mu¨ller et al. 2001, 2003, West et al. 2005) PCR method was used to isolate cDNA for these coding regions using the plastid and nucleus-encoded small subunit (H. S. Yoon et al. unpublished data). The PCR products were purified using the QIAquick PCR Purification Kit (Qiagen), (SSU) rDNA and plastid rbcL show furthermore that and were used for direct sequencing using the BigDyet Ter- the Porphyridiales are paraphyletic and comprise at minator Cycle Sequencing Kit (PE-Applied Biosystems, Nor- least three independent lineages. This result is gener- walk, CT, USA), and an ABI-3700 at the Roy J. Carver Center ally consistent with previous morphological studies for Comparative Genomics at the University of Iowa. Some that show great differences in plastid ultrastructure PCR products were cloned into the pGEM-T vector (Promega, and variable vegetative and reproductive anatomy Madison, WI, USA) before sequencing. (Gabrielson et al. 1985, 1990, Garbary and Gabrielson Phylogenetic analyses. We primarily used amino acid se- quences in the phylogenetic analysis in order to minimize 1990, Mu¨ller et al. 2001). However, all of these previ- potentially misleading phylogenetic signal because of DNA ous studies are characterized either by broad taxon mutation bias at the third positions of codons (Sanderson sampling with a single gene (Mu¨ller et al. 2001) or et al. 2000, Pinto et al. 2003) or because of heterogeneous narrow sampling with multiple genes (Yoon et al. codon usage (Inagaki et al. 2004). The protein sequences 2002b, 2004). For this reason, whereas the identity of were manually aligned using SeqPup (Gilbert 1995). Two the major red algal lineages has been relatively firmly data sets were used in the phylogenetic analyses and the alignments are available from D. Bhattacharya. In the first established, their interrelationships remain unclear. data set, we generated a concatenated alignment of seven Recently, Saunders and Hommersand (2004) pro- plastid-encoded proteins (7PEP; a total of 2564 aa): PsaA posed a new red algal taxonomic scheme based on (465 aa), PSI P700 chlorophyll a apoprotein A2 (PsaB, 422 previous molecular phylogenies and ultrastructural aa), PSII reaction center protein D1 (PsbA, 319 aa), PSII 44 characters (e.g. Golgi–ER association). Their taxo- KD apoprotein (PsbC, 334 aa), PSII D2 reaction center pro- nomic system is a large step forward but still contains tein (PsbD, 296 aa), RbcL (405 aa), and TufA (323 aa), from 16 bangiophytes, and from 15 outgroup taxa including green the paraphyletic class Rhodellophyceae that includes and glaucophyte algae and cyanobacteria. Because the rbcL both unicellular and pseudofilamentous forms (i.e. gene of the green and glaucophyte algae are of a cyanobac- Porphyridiales Kylin ex Skuja 1939, Stylonematales terial origin, whereas those in the red algae and red algal- K. Drew 1956, and ‘‘Porphyridiales 1’’ sensu Mu¨ller derived plastids are of proteobacterial origin (Valentin and et al. 2001). Zetsche 1990, Delwiche and Palmer 1996), the evolutionarily In this study, we determined 48 sequences from the distantly related green and glaucophyte